1. Technical Field
The present invention is directed to isolation and culture of adrenal medullary endothelial cells. More particularly, the present invention is related to a method of isolating and culturing endothelial cells of adrenal medullary origin which produce blood clotting Factor VIII:C.
2. Prior Art
Factor VIII is a blood protein and a component of the normal human blood. It is generally recognized that Factor VIII has at least three different functional sub units: (a) Factor VIII antigen (VIII AGN); (b) Blood clotting factor (VIII:C) that corrects the blood coagulation abnormality in classic hemophilia (hemophilia A) sometimes also designated as (VIII:AHF); and (c) von Willebrand Factor (VIII: vWF or VIII:R). As used herein the term Factor VIII is defined as and specifically refers to only VIII:C, the anti-haemophilic blood clotting factor and none other, except when otherwise noted. The significance of this blood clotting Factor VIII, of course, can be easily understood from the fact that its deficiency results in a bleeding disease commonly known as hemophilia. Blood clotting Factor VIII is, therefore, required for the treatment of hemophilia.
Factor VIII: C is quite distinct from other blood protein components. As mentioned supra, Factor VIII deficiency is associated with classic hemophilia A and von Willebrand's disease. Without being bound to any theory, it is hypothesized that two separate proteins may be involved. One is FVIII/vWF protein which is a glycoprotein that is multimeric, with molecular weights ranging from 850,000 to 30 million. The second protein is a 200,000 MW glycoprotein noncovalently complexed to the FVIII/vWF protein. It has procoagulant activity, termed VIII:C. The function of FVIII/vWF is to promote adhesion of platelets with the collagen coat associated with endothelial cells. It is controlled by a gene on the X-chromosome. The drug ristocetin causes platelets to aggregate by interacting with FVIII/vWF protein attached to platelets. In this guise it is also called FVIII:R or FVIII:RCoF. "RCoF" stands for ristocetin cofactor. An additional function of FVIII:R (or FVIII/vWF) may be to stabilize circulating FVIII:C for elaboration of its activity. The antibody usually raised against crude Factor VIII preparations is against FVIII:R, also termed Factor VIII-related antigen in the immunologically oriented literature. The site of synthesis of these factors is still not precisely known. The protein that resembles FVIII:R (factor VIII-related antigen, FVIII/vWF) has been shown to be synthesized by endothelial cells. The procoagulant component of factor VIII comes from an yet unidentified source (Fundamentals of Clinical Hematology, ed. by J. L. Spivak, Harper and Row Publ., Philadelphia, 1984, pages 367-372).
Heretofore, Factor VIII has been conventionally obtained from blood concentrates that may contain the pooled plasma of up to 20,000 blood donors. In accordance with the presently known process of obtaining therapeutic amounts of Factor VIII, pooled human serum or plasma is first treated by freezing and a cryoprecipitate is prepared therefrom. After thawing, Factor VIII is then precipitated by centrifugation. The precipitate is then used as a therapeutic agent or drug.
In accordance with a recent report, it has been estimated that "Although annual production of Factor VIII concentrates is 900 million international units (IU), production of cryoprecipitate, a substance rich in Factor VIII, is less than 300 million IU. These current supplies are clearly insufficient for the world's needs". (American Red Cross, Blood Services Bulletin, Vol. 17, No. 9, page 1, Aug. 15, 1984.)
In addition, among the various disadvantages and limitations of the currently used plasma process for preparing Factor VIII, at least the following should be noted.
(1) Almost inevitable presence of hepatitis viruses in the pooled blood concentrate as a result of which many hemophiliacs suffer from liver dysfunction caused by non-A and non-B hepatitis when Factor VIII prepared from such blood concentrates is administered to hemophiliac patients.
(2) High risk of causing Acquired Immune Deficiency Syndrome (AIDS) in patients treated with Factor VIII obtained from plasma concentrate because of potentially certain contamination of such plasma with AIDS virus.
(3) Need for human donors of blood, therefore, limited availability of Factor VIII.
(4) Human Factor VIII is contraindicated for some hemophiliacs due to such persons' developing antibodies against Factor VIII; therefore, Factor VIII from non-human sources has been considered to be of particular value for treatment of such hemophiliacs.
Factor VIII:C of the present invention, described fully hereunder, is believed to be of great significance in treating blood clotting abnormality in humans without the fear or risks associated with the conventional plasma preparations.
It has been known that Factor VIII is localized in, hence a marker of, endothelial cells (Hoyer et al., 1973, J. Clin, Invest. 52:2737-2744). However, endothelial cells originating from different tissues have different, tissue-specific biochemical and endocrine functions. For instance, capillary endothelial cells in adipose tissue may be involved in specific lipolytic reactions (Scow et al, 1976, Circ. Res. 39:149-162), while those in the smooth muscle may synthesize and secrete potent smooth muscle relaxing factors affecting neurotransmitter receptors (Smith et al., 1972, Handbook of Exp. Pharmacol, 33:538-605). In the lung, on the other hand, the function of the endothelial cells may include lipid metabolism leading to prostaglandin and prostacyclin synthesis, selective metabolism of biogenic amines and other vasoactive substances such as angiotensin and the metabolism of platelet aggregating agent adenosine diphosphate.
Although endothelial cells from other tissues have been studied, endothelial cells from adrenal medulla have not heretofore been isolated and cultured. A problem encountered is that most endothelial cells grow so slowly in vitro that they are not of any significant value from a practical stand point. Furthermore, they usually are capable of only up to 30 cycles of division and then die.
The present invention for the first time provides a unique line of endothelial cells capable of continuous growth and a method of isolating and culturing in vitro said endothelial cells of adrenal medullary origin, which multiply profusely and produce blood clotting Factor VIII:C having none of the disadvantages or limitations of the conventionally used blood concentrate FVIII:C. The invention also provides for the first time the opportunity of studying various properties of endothelial cells including structural, physiological and biochemical attributes thereof.